A Novel Droplet Digital PCR Method for Population-based Plasmodium falciparum hrp2 Deletion Diagnosis Project Summary Malaria is a major cause of morbidity and mortality in the tropics and subtropics. Malaria treatment, control and elimination is a top public health priority. Diagnosis of malaria parasites in febrile cases in order to administer appropriate treatment is a major challenge. Current rapid diagnostic tests (RDTs) that rely on detecting proteins secreted by malaria parasite in the human blood represent a crucial tool for malaria diagnosis in the field, particularly in resource-poor areas where microscopes and electricity are lacking. Over 300 million RDTs were sold in 2014 worldwide, and over two thirds of malaria diagnosis in Africa were based on RDT. The most sensitive RDTs to diagnose Plasmodium falciparum ? the deadliest malaria parasite ? rely on detection of the Histidine Rich Protein 2 (HRP2). Yet, several studies have documented hrp2 gene deletion in the parasite, rendering RDTs unable to detect these parasites. Available protocols to screen for hrp2 deletion require a series of PCR, facing an inherent risk of false negative results. Here, we propose to develop a novel and rapid assay to screen for parasite gene deletions using droplet digital PCR (ddPCR). The preliminary studies found that this method is highly sensitive, minimizes the risk of false-negative results, and can detect mixed infections with hrp2-delection and wildtype parasites. The objective of this application is to: 1) develop and validate the ddPCR method for detecting hrp2 deletion, and 2) test the hypothesis that the frequency of hrp2- negative P. falciparum parasites has increased due to the selection pressure of HRP2-based diagnosis, and that hrp2-negative P. falciparum parasites are widespread. A unique aspect of this project is the linkage with study sites under NIH-funded International Center of Excellence in Malaria Research (ICEMR) and samples from Africa, Asia, the South Pacific and Latin America will be analyzed. The outcome of this application includes: 1) a rapid and sensitive hrp2 deletion detection method, and 2) understanding the extent of hrp2 deletion in a large multi-continental scale. This knowledge will inform whether the current RDT diagnosis technique grossly misdiagnoses malaria infections and where complementary diagnostic methods should be used.